Ether-linked glycerolipids are characteristically elevated in most cancer cells. Their biological significance recently surfaced when certain molecular species of ether lipids were shown to possess potent and diverse biological activities. During the past year a major accomplishment occurred in our enzymatic mechanistic studies of alkyldihydroxyacetone-P (alkyl-DHAP) synthase, the enzyme that catalyzes the formation of the ether bond in lipids. Using Ehrlich ascites cell membranes as the source, we were able to solubilize and purify this enzyme more than 1,000-fold. This achievement permitted for the first time a detailed study of the reaction mechanism of alkyl-DHAP synthase in a preparation that was completely free of all other enzymes that compete for the substrates. Kinetic analysis established the order of addition of substrates and the release of products; the results have also revealed the existence of an enzyme-DHAP intermediate in the ether bond-forming reaction. These experiments have provided strong evidence to support the concept that the reaction catalyzed by alkyl-DHAP synthase occurs via a ping-pong mechanism. Intermediary binary complexes are formed instead of ternary complexes as in a sequential mechanism previously proposed by others who had used intact microsomes as the enzyme source. Knowing the reaction mechanism of the ether bond-forming reaction could lead to the development of new inhibitors for this pathway of membrane lipids in cancer cells. Also, the purified alkyl-DHAP synthase preparation is expected to yield an antibody that can be used in regulatory studies of this very important enzyme.